Peptidases (also referred to as proteinases or proteases) are enzymes which catalyze the cleavage of peptide bonds in peptides or proteins. In the form of endopeptidases, they prefer as reactants peptide bonds within the target molecule which generally involve specific amino acids. Other peptidases cleave individual or several amino acids from the N- or C-terminus of the protein. Depending on their tertiary structure, the different endopeptidases are often able to react with different physiological or unphysiological reactants of different molecular size comprising different specific peptide bonds.
Peptidases fulfill important functions in the organism and are involved in the physiological, but also pathological, procedures of numerous processes such as e.g., the breakdown of proteins, immune response (complement system), blood coagulation and fibrinolysis.
In addition, in some cases the activated endopeptidases also form macromolecular complexes with cofactors, such as e.g., the activated coagulation factor Xa with factor Va in the prothrombinase complex.
However, for certain diagnostic as well as therapeutic processes, this spectrum of reactants is disadvantageous for a purposeful, specific use of such enzymes.
The process of blood coagulation is very complex and involves several steps whereby inactive enzyme precursors are activated by a combination of enzymatic action and cofactors which results in active coagulation enzymes.
For balancing the equilibrium between the coagulation tendency of the blood and safeguarding its flow properties within the circulation, there is also another interconnected system of inhibiting or amplifying interactions with additional molecules which is regulated by numerous positive and negative feedback mechanisms.
New classes of drugs have been and are being developed for the treatment of defects of the blood coagulation system, which specifically only inhibit a single enzyme of the coagulation cascade, such as e.g., the activated factor Xa (active ingredient class of direct factor Xa inhibitors e.g., Rivaroxaban®, Apixaban®, Betrixaban®, Otamixaban®, Edoxaban®, Eribaxaban, YM150, LY-517717, PRT054021 and others) or thrombin (active ingredient class of direct thrombin inhibitors, e.g., Dabigatran®, Argatroban, Bivalirudin, MCC-977, AZD0837, NU172, Flovagatran and others).
In order to guarantee an effective (prevention of thrombosis) and safe therapy (prevention of bleeding) with such drugs, both the availability of exact methods for detecting the active ingredient in bodily fluids as well as the possibility of neutralizing or removing these drugs are of the utmost importance.
In order to be able to purposefully measure or neutralize drugs in vitro or in vivo which inhibit specific enzymes of the hemostatic system, it would be highly advantageous if the spectrum of possible reaction of the various enzymes could be modified or limited.
Accordingly, modifications of the molecular structure of thrombin and factor Xa have become known in the course of the development of antidotes to factor Xa and thrombin inhibitors which cause these peptidases to lose their ability to function and interact in the coagulation system while continuing to interact with the specific inhibitor substances. Such modified peptidases are intended to be used to neutralize the active ingredient. “Neutralization” herein refers to a binding of the active ingredient molecule to the peptidase which leads to the inability of the active ingredient molecule to perform its function as an inhibitor of the hemostatic system. This may be achieved by removing and/or exchanging specific amino acids. Documents U.S. Pat. No. 6,060,300 (Thrombin muteins as antidotes for thrombin inhibitors) and WO 2009/041962 (Antidotes for Factor Xa inhibitors and methods of using the same), inter alia, disclose intricate processes for this purpose. In addition, US 2009/0098119A1 discloses that the inherently short half life of the mutants of factor Xa can be extended by a reaction with polyethylene glycol, but not that other properties of the mutated factor Xa molecule are changed due to this modification.